JP2016109666A - Insulin detecting method and insulin detecting kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an insulin detecting method and insulin detecting kit which can conveniently detect insulin.SOLUTION: Insulin is detected by detecting fluorescence generated from luminescence resonance energy transfer between a light emitting material or fluorescence material coupled to an α-CT segment of insulin receptor, and a fluorescence material or light emitting material coupled to an L1 domain of insulin receptor.SELECTED DRAWING: None

Description

  The present invention relates to an insulin detection method and an insulin detection kit.

  Insulin is a peptide hormone that controls the blood glucose level (blood glucose level). When insulin is secreted from β-cells of the pancreas in response to a blood glucose level that rises after a meal, muscle cells, fat cells, and the like sense insulin, and these cells absorb glucose. As a result, the blood sugar level decreases.

  Much research has been conducted on the signal transduction mechanism of insulin in vivo, and various things have been clarified regarding the structure of insulin and the structure of the insulin receptor to which insulin binds.

  Insulin receptors are proteins that contain two α subunits and two β subunits. More specifically, each of the α subunits is a region disposed outside the cell and a region to which insulin binds. Each of the α subunits also contains an α-CT segment and an L1 domain, and these two domains bind to insulin. On the other hand, each of the β subunits is a region that is arranged inside the cell, and is a region that generates various signals in the cell when insulin is bound to the α subunit. In addition, each of the β subunits includes a transmembrane region and a part of the extracellular domain, and the insulin receptor can exist as a membrane protein in the cell by the action of the transmembrane region ( For example, refer nonpatent literature 1 and FIG.

  A diabetic patient is in a state where the blood sugar level cannot be sufficiently controlled by decreasing the secreted amount of insulin or reducing the effect of insulin. Therefore, the development of a technique for detecting insulin and a technique for measuring the concentration of insulin are underway for the development of a therapeutic method for diabetes and for the maintenance of health.

  For example, as such a technique, a technique using ELISA (Enzyme-Linked ImmunoSorbent Assay) can be cited (for example, see Non-Patent Documents 2 to 4). However, the technique using ELISA has many problems such as complicated operations such as washing and the need to produce antibodies. High-throughput detection in drug development, POCT ( It is not suitable for Point of Care Testing. In addition, a homogeneous assay method that does not require an operation such as washing has been developed. However, in this method, it is necessary to chemically bind the anti-insulin antibody and the signal production site in advance and purify the conjugate. I have a problem. Therefore, development of simpler technology is required.

  Under such circumstances, activation of the insulin receptor using LRET (luminescence resonance energy transfer), more specifically, BRET (Bioluminescence Resonance Energy Transfer). Development of a technique for detecting (in other words, the binding between an insulin receptor and insulin) is underway (see, for example, non-patent documents 5 to 7).

  The techniques described in Non-Patent Documents 5 to 7 use a complete form of an insulin receptor having two α subunits and two β subunits. One β subunit is linked to RLuc (Renilla Luciferase), the other β subunit is linked to YFP (Yellow Fluorescent Protein), and when insulin is bound to the α subunit, RLuc, YFP, Luminescence resonance energy transfer occurs between them, and as a result, YFP fluoresces. Then, activation of the insulin receptor (in other words, binding between the insulin receptor and insulin) is detected by detecting the fluorescence.

John G. Menting et al., “How insulin engages its primary binding site on the insulin receptor” Nature, 10 January 2013, Vol.493, p241-245 Makoto Shigeto et. Al., “First Phase of Glucose-Stimulated Insulin Secretion From MIN6 Cells Does Not Always Require Extracellular Calcium Influx” J Pharmacol Sci 101, p293-302 (2006) Eiji Yamato et. Al., “Microarray Analysis of Novel Candidate Genes Responsible for Glucose-Stimulated Insulin Secretion in Mouse Pancreatic β Cell Line MIN6” PLOS ONE / 1439181894901_0.org April 2013, Vol.8, Issue.4, e61211 Miyazaki J. et. Al., “Establishment of a Pancreatic β Cell Line That Retains Glucose-Inducible Insulin Secretion: Special Reference to Expression of Glucose Transporter Isoforms., MIN6 cell line” Endocrinology, 127, p126-132 (1990) Nicolas Boute et. Al., “Monitoring the Activation State of the Insulin Receptor Using Bioluminescence Resonance Energy Transfer” Molecular Pharmacology, Vol.60, No.4, p640-645 (2001) Tarik Issad et. Al., “A homogenous assay to monitor the activity of the insulin receptor using Bioluminescence Resonance Energy Transfer” Biochemical Pharmacology, 64, p813-817 (2002) Christophe Blanquart et.al., “Monitoring the Activation State of the Insulin-Like Growth Factor-1 Receptor and its Interaction with Protein Tyrosine Phosphatase 1B Using Bioluminescence Resonance Energy Transfer” Molecular Pharmacology, Vol.68, No.3, p885-894 (2005)

  However, the conventional techniques described in Non-Patent Documents 5 to 7 described above have a problem that insulin cannot be easily detected.

  Specifically, the conventional techniques described in Non-Patent Documents 5 to 7 use a complete form of an insulin receptor, in other words, an insulin receptor that is a membrane protein. Therefore, the conventional techniques described in Non-Patent Documents 5 to 7 have the following problems: (I) a complicated operation for purifying an insulin receptor that is a membrane protein; and (II) an insulin receptor that is a membrane protein. However, the insulin receptor, which is a membrane protein, is easily denatured and difficult to handle.

  The present invention has been made in view of the above-described conventional problems, and an object thereof is to provide an insulin detection method and an insulin detection kit capable of easily detecting insulin.

  In order to solve the above-described problem, the insulin detection method of the present invention includes a sample, a luminescent substance, and a first polypeptide that contains an α-CT segment of an insulin receptor and does not contain a β subunit of the insulin receptor. Alternatively, a first complex in which a fluorescent substance is linked and a second polypeptide that contains the L1 domain of the insulin receptor and does not contain the β subunit of the insulin receptor is linked to a luminescent substance or a fluorescent substance. And a fluorescence detection step of detecting fluorescence generated by luminescence resonance energy transfer generated between the luminescent substance and the fluorescent substance from a solution containing the complex of two, wherein the luminescent substance is present in the first polypeptide. When linked, a fluorescent substance is linked to the second polypeptide, and a fluorescence is linked to the first polypeptide. If the quality is coupled is characterized in that the luminescent substance to the second polypeptide is linked.

  In the insulin detection method of the present invention, the concentration of the first complex (M1) and the concentration of the second complex (M2) in the solution have a relationship of 50 ≦ M2 / M1 ≦ 200. It is preferable to satisfy.

  In the method for detecting insulin of the present invention, the first complex is preferably one in which the luminescent substance or the fluorescent substance is linked to the amino terminus of the first polypeptide.

  In the method for detecting insulin of the present invention, the second complex is preferably one in which the luminescent substance or the fluorescent substance is linked to the carboxyl terminus of the second polypeptide.

  In the method for detecting insulin of the present invention, it is preferable that the first complex and the second complex are bonded to each other by a linker to form a third complex.

  In the insulin detection method of the present invention, the third complex is preferably bound to the surface of the support.

  In order to solve the above-described problem, the insulin detection kit of the present invention contains a luminescent substance or a fluorescent substance in the first polypeptide containing the α-CT segment of the insulin receptor and not containing the β subunit of the insulin receptor. And a second complex comprising a luminescent substance or a fluorescent substance linked to a second polypeptide containing the L1 domain of the insulin receptor and not containing the β subunit of the insulin receptor. The luminescent substance and the fluorescent substance generate fluorescence by luminescence resonance energy transfer generated between the luminescent substance and the fluorescent substance, and emit light to the first polypeptide. When a substance is linked, a fluorescent substance is linked to the second polypeptide, and the first polypeptide is linked. When the fluorescent substance is coupled to the tides, it is characterized in that the luminescent substance to the second polypeptide is linked.

  In the insulin detection kit of the present invention, the first complex and the second complex are preferably bonded to each other by a linker to form a third complex.

  The insulin detection kit of the present invention preferably includes a support on which the third complex is bound to the surface.

  The present invention does not require complicated operations such as an antibody production operation, a membrane protein purification operation, and a membrane protein storage operation that is easily denatured, and therefore has the effect that insulin can be easily detected.

  The first complex and the second complex used in the present invention can be purified with high purity. Therefore, the present invention has an effect that insulin can be detected in a state where there are few impurities. That is, the present invention has an effect that insulin can be detected without being affected by impurities.

It is the dyeing | staining image of the polyacrylamide gel after SDS-PAGE in the Example of this invention. In the Example of this invention, it is the graph which plotted the value of BRET efficiency under fixed insulin density | concentration at the time of changing the ratio of the quantity of the 1st complex, and the quantity of the 2nd complex. In the Example of this invention, it is the graph which plotted the value of BRET efficiency with respect to the value of insulin concentration. In the Example of this invention, it is a graph which shows the change of BRET efficiency at the time of making insulin secret from a biological body. It is a graph which shows the measurement result of the fluorescence generate | occur | produced by the luminescence resonance energy transfer in the Example of this invention. It is a graph which shows the measurement result of the fluorescence generate | occur | produced by the luminescence resonance energy transfer in the Example of this invention. It is a graph which shows the measurement result of the fluorescence generate | occur | produced by the luminescence resonance energy transfer in the Example of this invention. It is a figure which shows the outline of the structure of an insulin receptor. (A) is the dyeing | staining image of the polyacrylamide gel after isolate | separating the sample before a refinement | purification in SDS-PAGE in the Example of this invention, (b) is the after-purification in the Example of this invention. It is the dyeing | staining image of the polyacrylamide gel after isolate | separating a sample by SDS-PAGE. It is a graph which shows the measurement result of the fluorescence generate | occur | produced by the luminescence resonance energy transfer in the Example of this invention. (A) And (b) is the graph which plotted the value of BRET efficiency with respect to the value of insulin concentration in the Example of this invention. It is a graph which shows the measurement result of the fluorescence generate | occur | produced by the luminescence resonance energy transfer in the Example of this invention. It is a graph which shows the measurement result of the fluorescence generate | occur | produced by the luminescence resonance energy transfer in the Example of this invention. It is an image of co-culture of sensor cells and insulin-secreting cells in an Example of the present invention. It is image data which shows the change of BRET efficiency which a sensor cell shows in the Example of this invention when insulin does not exist. It is image data which shows the change of BRET efficiency which a sensor cell shows in the Example of this invention when insulin exists. (A) shows the image data of the sensor cell when insulin is not present and the region used for calculating the average value of BRET efficiency in the embodiment of the present invention, and (b) shows the region in the embodiment of the present invention. FIG. 4 is a graph showing the change in the average value of the BRET efficiency in the region indicated by the sensor cell when insulin is not present, in which (c) is a sensor cell when insulin is present according to an embodiment of the present invention. (D) is the average value of the BRET efficiency in the above-mentioned region indicated by the sensor cell when insulin is present in the example of the present invention. It is the graph which digitized the change of.

  An embodiment of the present invention will be described below, but the present invention is not limited to this. The present invention is not limited to each configuration described below, and various modifications can be made within the scope shown in the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are used as references in this specification. Unless otherwise specified in this specification, “A to B” representing a numerical range is intended to be “A or more and B or less”.

[1. (Insulin detection method)
In the method for detecting insulin according to the present embodiment, a luminescent substance or a fluorescent substance is linked to a first polypeptide that contains a sample and an α-CT segment of the insulin receptor and does not contain the β subunit of the insulin receptor. A second complex in which a luminescent substance or a fluorescent substance is linked to a second polypeptide containing the L1 domain of the insulin receptor and not containing the β subunit of the insulin receptor; A fluorescence detecting step of detecting fluorescence generated by luminescence resonance energy transfer generated between the luminescent substance and the fluorescent substance from a solution containing the luminescent substance. Each configuration will be described below.

[1-1. sample〕
The sample is a test subject that is tested for insulin and / or to what extent insulin is tested. Therefore, the method for detecting insulin of the present embodiment can be a method for quantifying insulin.

  If the sample contains insulin, the α-CT segment and the L1 domain form a complex via the insulin. If the α-CT segment and the L1 domain form a complex, the luminescent substance (or fluorescent substance) linked to the α-CT segment and the fluorescent substance (or luminescent substance) linked to the L1 domain Will approach. When the luminescent substance and the fluorescent substance come close to each other, light is emitted from the luminescent substance toward the fluorescent substance, and luminescence resonance energy transfer occurs between the luminescent substance and the fluorescent substance. As a result, the fluorescent substance emits fluorescence. Occur. Then, by detecting the fluorescence, insulin contained in the sample can be detected and / or quantified.

  The specific configuration of the sample is not particularly limited, and may be an artificially prepared sample (for example, a drug such as a therapeutic drug for diabetes) or a sample collected from a living body.

[1-2. First complex]
In the first complex, a luminescent substance or a fluorescent substance is linked to a first polypeptide containing an α-CT segment of an insulin receptor and not containing a β subunit of the insulin receptor.

  In a second complex described later, when a luminescent substance is linked to the second polypeptide, a fluorescent substance is linked to the first polypeptide in the first complex. On the other hand, when a fluorescent substance is linked to the second polypeptide in the second complex, a luminescent substance is linked to the first polypeptide in the first complex. With the above configuration, when the α-CT segment and the L1 domain form a complex, the light-emitting substance and the fluorescent substance can be approached, so that fluorescence can be generated efficiently.

  The first polypeptide that is a component of the first complex is a polypeptide containing the α-CT segment of the insulin receptor. The α-CT segment is a partial region of the α subunit that is the extracellular domain of the insulin receptor, has the ability to bind to insulin, and does not contain a transmembrane region. In addition, the specific structure of the α-CT segment in the present invention is not particularly limited, and may be any known α-CT segment.

  The first polypeptide that is a constituent component of the first complex is a polypeptide that does not contain the β subunit of the insulin receptor. The β subunit is the intracellular domain of the insulin receptor and includes a transmembrane region and a part of the extracellular domain. The insulin receptor can exist as a membrane protein in the cell by the action of the transmembrane region. The specific configuration of the β subunit in the present invention is not particularly limited, and may be any known β subunit.

  As described above, the first polypeptide does not include a transmembrane region. In other words, the first complex does not include a transmembrane region. Therefore, since the first complex can be a soluble protein (in other words, a non-membrane protein), it can be easily purified with high purity, is easy to handle, and easily contains insulin and the second protein. A complex can be formed with the complex.

  In the present specification, the transmembrane region means a partial region in the membrane protein that is arranged so as to be embedded inside the cell membrane when the membrane protein is arranged on the cell membrane. Many transmembrane regions have a structure formed by a hydrophobic amino acid, more specifically, an α-helix structure formed by a hydrophobic amino acid. It is not limited to the structure.

  The first polypeptide, which is a constituent component of the first complex, may be composed of the α-CT segment of the insulin receptor, or may include components other than the α-CT segment of the insulin receptor. May be.

  When the first polypeptide includes a configuration other than the α-CT segment of the insulin receptor, the configuration may be any configuration as long as it does not include a transmembrane region. As the said structure, the linker which connects an alpha-CT segment, and a luminescent substance or a fluorescent substance may be sufficient, for example. The linker may be a peptidic linker or a non-peptidic linker (for example, a crosslinking agent).

  When the luminescent substance or fluorescent substance is linked to the first polypeptide, the luminescent substance or fluorescent substance may be linked to the amino terminus of the first polypeptide, or the luminescent substance is linked to the carboxyl terminus of the first polypeptide. Alternatively, a fluorescent substance may be linked.

  From the viewpoint of stabilizing the structure of the first complex and performing better insulin detection, the first complex has a luminescent substance or fluorescent substance linked to the amino terminus of the first polypeptide. It is preferable that If it is the said structure, the distance between the luminescent substance and fluorescent substance which are contained in a 1st composite_body | complex and a 2nd composite_body | complex can be shortened, and the luminescence resonance energy transfer can be efficiently caused.

  More specifically, when a luminescent substance is linked to the amino terminus of the first polypeptide, it is preferable to link a fluorescent substance to the carboxyl terminus of the second polypeptide described later, When a fluorescent substance is linked to the amino terminus, it is preferable to link a luminescent substance to the carboxyl terminus of the second polypeptide described below.

  The luminescent substance and fluorescent substance to be linked to the first polypeptide are not particularly limited as long as they can generate fluorescence by luminescence resonance energy transfer, and are preferably a luminescent protein and a fluorescent protein, respectively. With this configuration, the first complex can be formed as one fusion protein (specifically, a fusion protein of the first polypeptide and a luminescent protein or a fluorescent protein). A first complex can be easily prepared by introducing an expression vector into which a polynucleotide encoding a fusion protein is inserted into a desired host and expressing the fusion protein in the host. Furthermore, if it is the said structure, the operation which connects a 1st polypeptide, and a luminescent substance or a fluorescent substance through a chemical bond, and the operation | movement which refine | purifies the 1st complex after connecting are made unnecessary. Can do.

  The luminescent substance in this specification may be any substance involved in the luminescence phenomenon. For example, when light is emitted by the reaction between an enzyme and a substrate, the luminescent substance may be an enzyme or a substrate of the enzyme. From the viewpoint of better detecting insulin, the luminescent substance is preferably an enzyme.

  Examples of the luminescent substance include luciferase. Examples of luciferase include, for example, firefly-derived luciferase, Renilla luciferase, and luciferase derived from Oplophorus gracilirostris. Examples of the luciferase derived from shrimp are: NanoLuc (Nluc) (registered trademark), terbium chelate diethylenetriaminepentacetate-carbostyril 124-maleimidopropionic hydrazide (Tb-DTPA-cs124-EMPH) (for example, Elise Burmeister Getz et al., Biophysical Journal, Volume 74, May 1998, p2451-2458).

  Examples of fluorescent substances include, for example, well-known fluorescent proteins (for example, Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, Midorishi Cyan, CFP, TurboGFP, AcGFP, TagGFP, Azami-P, Azami-P , GFP2, HyPer, TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana, KusabiraOrange, mOrange, TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, TurboFP602, mRF 1, JRed, KillerRed, mCherry, HcRed, KeimaRed, mRasberry, mPlum, PS-CFP, Dendra2, Kaede, EosFP, and can include KikumeGR).

  Examples of the fluorescent material include TMR, diAcFAM, and Oregon green. These fluorescent materials can generate fluorescence by luminescence resonance energy transfer that occurs with NanoLuc (Nluc) (registered trademark). Examples of the fluorescent material include Cy3 and Cy3.5. These fluorescent substances can generate fluorescence by luminescence resonance energy transfer that occurs with firefly-derived luciferase.

[1-3. Second complex]
In the second complex, a luminescent substance or a fluorescent substance is linked to a second polypeptide that contains the L1 domain of the insulin receptor and does not contain the β subunit of the insulin receptor.

  In the first complex described above, when a fluorescent substance is linked to the first polypeptide, a luminescent substance is linked to the second polypeptide in the second complex. On the other hand, when a luminescent substance is linked to the first polypeptide in the first complex, a fluorescent substance is linked to the second polypeptide in the second complex. With the above configuration, when the α-CT segment and the L1 domain form a complex, the light-emitting substance and the fluorescent substance can be approached, so that fluorescence can be generated efficiently.

  The second polypeptide that is a constituent component of the second complex is a polypeptide containing the L1 domain of the insulin receptor. The L1 domain is a partial region of the α subunit that is the extracellular domain of the insulin receptor, has the ability to bind to insulin, and does not include a transmembrane region. In addition, the specific structure of the L1 domain in the present invention is not particularly limited, and may be any known L1 domain.

  The second polypeptide that is a constituent component of the second complex is a polypeptide that does not contain the β subunit of the insulin receptor. The β subunit is the intracellular domain of the insulin receptor and includes a transmembrane region and a part of the extracellular domain. The insulin receptor can exist as a membrane protein in the cell by the action of the transmembrane region. The specific configuration of the β subunit in the present invention is not particularly limited, and may be any known β subunit.

  As described above, the second polypeptide does not include a transmembrane region. In other words, the second complex does not include a transmembrane region. Therefore, since the second complex can be a soluble protein (in other words, a non-membrane protein), it can be easily purified with high purity, is easy to handle, and easily contains insulin and the first complex. A complex can be formed with the complex.

  The second polypeptide that is a constituent component of the second complex may be composed of the L1 domain of the insulin receptor, or may include a component other than the L1 domain of the insulin receptor.

  When the second polypeptide includes a configuration other than the L1 domain of the insulin receptor, the configuration may be any configuration as long as it does not include a transmembrane region. As the said structure, the linker which connects L1 domain, and a luminescent substance or a fluorescent substance may be sufficient, for example. The linker may be a peptidic linker or a non-peptidic linker (for example, a crosslinking agent).

  When the luminescent substance or fluorescent substance is linked to the second polypeptide, the luminescent substance or fluorescent substance may be linked to the amino terminus of the second polypeptide, or the luminescent substance is linked to the carboxyl terminus of the second polypeptide. Alternatively, a fluorescent substance may be linked.

  From the standpoint of better insulin detection by stabilizing the structure of the second complex, the second complex has a luminescent or fluorescent substance linked to the carboxyl terminus of the second polypeptide. It is preferable. If it is the said structure, the distance between the luminescent substance and fluorescent substance which are contained in a 1st composite_body | complex and a 2nd composite_body | complex can be shortened, and a luminescence resonance energy transfer can be raise | generated efficiently.

  More specifically, when a luminescent substance is linked to the carboxyl terminus of the second polypeptide, it is preferable to link a fluorescent substance to the amino terminus of the first polypeptide described above. When a fluorescent substance is linked to the carboxyl terminus, it is preferable to link a luminescent substance to the amino terminus of the first polypeptide.

  The luminescent substance and fluorescent substance to be linked to the second polypeptide are not particularly limited as long as they can generate fluorescence by luminescence resonance energy transfer, and are preferably a luminescent protein and a fluorescent protein, respectively. With this configuration, the second complex can be formed as one fusion protein (specifically, a fusion protein of the second polypeptide and a luminescent protein or a fluorescent protein). A second complex can be easily prepared by introducing an expression vector into which a polynucleotide encoding the fusion protein is inserted into a desired host and expressing the fusion protein in the host.

  As the luminescent substance and fluorescent substance to be linked to the second polypeptide, the same substances as those described as the luminescent substance and fluorescent substance that can be linked to the first polypeptide can be used. Since specific examples of these light-emitting substances and fluorescent substances have already been described, description thereof is omitted here.

[1-4. Example of Embodiment of First Complex and Second Complex]
The first complex and the second complex may be separated from each other, but may be combined with each other by a linker to form a third complex. .

  If the first complex and the second complex are bound to each other by a linker, the first complex and the second complex (in other words, the α-CT segment and the L1 domain) There can always be nearby. In this case, for example, if insulin binds to the first complex containing the α-CT segment, the second complex containing the L1 domain can also be efficiently bound to the insulin. As a result, even when the insulin concentration is low, luminescence resonance energy transfer can be efficiently generated, and as a result, fluorescence can be efficiently generated from the fluorescent substance.

  In this case, the region in the first complex to which the linker is bonded and the region in the second complex to which the linker is bonded are not particularly limited. For example, (i) the luminescent substance or fluorescent substance in the first complex and the second polypeptide in the second complex may be linked by a linker, or (ii) the first complex in the first complex The luminescent substance or the fluorescent substance and the luminescent substance or the fluorescent substance in the second complex may be linked by a linker, or (iii) the first polypeptide in the first complex and the second complex The second polypeptide in the body may be bound by a linker, or (iv) the first polypeptide in the first complex and the luminescent substance or fluorescent substance in the second complex are linked to the linker. You may combine by. From the viewpoint of realizing the third composite having a more stable structure, (iii) is most preferable among the above (i) to (iv).

  The linker is not particularly limited as long as it can bind the first complex and the second complex. Examples of the linker include a polypeptide. If a polypeptide linker is used, the third complex can be configured as one protein (for example, a fusion protein of the first complex, the linker, and the second complex). And if it is the said structure, a fusion protein can be easily produced by producing the expression vector of the said fusion protein by a well-known method, and introduce | transducing the said expression vector into a desired host.

  When a polypeptide is used as the linker, the number of amino acids constituting the linker is not particularly limited. From the viewpoint of measuring a lower concentration of insulin, the number of amino acids constituting the linker is 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, or 26. It is preferable that they are 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, or 34 or more. In these cases, the upper limit of the number of amino acids constituting the linker is not particularly limited (for example, 100 or any integer of 100 or more).

  If the linker is too short (for example, the number of amino acids constituting the linker is 19 or less), the distance between the luminescent substance and the fluorescent substance is kept short even if insulin is not present. Emission resonance energy transfer occurs between them, and as a result, fluorescence is generated. That is, when the linker is too short, the intensity of background fluorescence when detecting insulin tends to increase. Therefore, from the viewpoint of detecting insulin with high sensitivity, the linker preferably has the above-described length.

  When using polypeptide as a linker, the kind of amino acid which comprises the said linker is not specifically limited. From the viewpoint of measuring a lower concentration of insulin, the linker is preferably composed of an amino acid having a high degree of freedom in steric structure. More specifically, the linker is (i) at least one amino acid selected from the group consisting of glycine, alanine, valine, leucine and isoleucine, which is an amino acid having a fatty acid side chain; an amino acid having a side chain containing a hydroxyl group And at least one amino acid selected from the group consisting of serine, threonine and tyrosine; and selected from the group consisting of aspartic acid, asparagine, glutamic acid and glutamine, which are amino acids having side chains including acids or amides At least one amino acid, or (ii) at least one amino acid selected from the group consisting of glycine, alanine and leucine, which is an amino acid having a fatty acid side chain; An amino acid having a side chain containing Phosphorus; and glutamic acid that is an amino acid having a side chain containing an acid, or (iii) may be composed of glycine, alanine, serine, leucine, and glutamic acid Alternatively, (iv) the polypeptide represented by “ASSAGGSAGGSAGGSAGGSAGGSAGGSAGSGGLE (SEQ ID NO: 14)” may be included as at least a part.

  The third complex can be bound to the surface of a support (eg, a glass substrate, bead, gel, cell, or extracellular matrix). If the first complex and the second complex are integrated as a third complex, the surface of the support is maintained while keeping the first complex and the second complex in a favorable arrangement with respect to each other. Can be combined. And if the said support body is used, insulin can be detected with sufficient sensitivity.

  The method for binding the third complex to the surface of the support is not particularly limited, and the third complex and the support may be bound by a cross-linking agent, or depending on the tag included in the third complex. The third complex and the support may be combined, or the third complex and the support may be combined with a tag included in the support.

  The method of binding the third complex to the surface of a cell that is one of the supports is not particularly limited. For example, the third complex may be bound to the surface of the cell by introducing an expression vector of the third complex into the cell and expressing the third complex in the cell. In this case, the third complex may be bound to the surface of the cell by the action of the tag described later, or the third complex may be bound to the surface of the cell without the action of the tag described later. Also good.

  Alternatively, the third complex may be bound to the cell surface by mixing the third complex and the cell. In this case, the third complex may be bound to the surface of the cell by the action of the tag described later, or the third complex may be bound to the surface of the cell without the action of the tag described later. Also good.

  As shown in the examples described below, the present inventor confirmed that (i) the third complex is structurally stable and can be expressed in a large amount in a desired cell, (ii) the third complex When a tag (specifically, a tag that binds to a cell membrane) is artificially bound to the complex, the third complex to which the tag is bound is not destabilized structurally, (Iii) the third complex to which the tag is bound is present on the surface of the cell by the action of the tag, and is produced by another cell in the vicinity of the cell. It was found that it can be detected. Therefore, a tag for binding to the cell membrane may be bound to the third complex.

  The tag may be any tag that binds to the cell membrane, and the specific configuration is not particularly limited. The tag is preferably a polypeptide. If a polypeptide tag is used, the third complex and the tag are configured as one protein (for example, a fusion protein of the first complex, the linker, the second complex, and the tag). Can do. And if it is the said structure, the fusion protein couple | bonded with the cell membrane of a host is easily produced by producing the expression vector of the said fusion protein by a well-known method, and introduce | transducing the said expression vector into a desired host. can do.

  Specific examples of tags include membrane proteins (eg, PDGF (platelet-derived growth factor) receptor, β2 adrenergic receptor (β2AR) (eg, John Holleran et al., Fluorogen-Activating Proteins as Biosensors of Cell-Surface Proteins in Living). Cells, Journal of the International Society for Advancement of Cytometry, Cytometry PART A, Vol. 77A, 776-782, 2010), EGF (Epidermal growth factor) receptor, insulin receptor, VGF (vascular endothelial cell growth factor) receptor, FGF (Fibroblast growth factor) receptor or DDR (discodin domain receptor) transmembrane region.

  The location where the tag in the third complex is bound is not particularly limited. In the case where the third complex and the tag are configured as one protein using a polypeptide tag, the amino terminus of the third complex is used from the viewpoint of further stabilizing the structure of the third complex. Alternatively, it is preferable to attach a tag to the carboxyl terminus.

[1-5. (Fluorescence detection process)
In the fluorescence detection step, light that decreases due to light emission resonance energy transfer that occurs between the luminescent substance and the fluorescent substance from a solution containing the sample, the first complex, and the second complex, and generated fluorescence It is a process of detecting.

  For example, if fluorescence is detected in the above-described fluorescence detection step (or if a decrease in luminous ability inherent in the luminescent substance and an increase in fluorescence are detected), insulin is contained in the sample Can be determined. On the other hand, if no fluorescence is detected in the above-described fluorescence detection step (or if the decrease in luminous ability inherent in the luminescent substance and the enhancement of fluorescence are not detected), the sample contains no insulin Can be determined.

  The wavelength of the fluorescence detected in the fluorescence detection step is not particularly limited, and can be set as appropriate according to the combination of the luminescent substance and the fluorescent substance.

  The fluorescence wavelength (peak wavelength: A (nm)) detected in the fluorescence detection step is preferably as far as possible from the wavelength (peak wavelength: B (nm)) of the light emitted by the luminescent material. For example, the absolute value of (AB) is preferably 50 or more, more preferably 60 or more, further preferably 70 or more, and most preferably 80 or more. The upper limit of the absolute value is not particularly limited, but may be 100 or 150 in consideration of the detection capability of the fluorescence detection device, for example. If it is the said structure, since each of the intensity | strength of the fluorescence detected and the intensity | strength of the light which a luminescent substance emits can be measured correctly, insulin can be detected more correctly.

  The concentration of the first complex (M1) and the concentration of the second complex (M2) in the solution are not particularly limited, but the first complex and the second complex are mutually connected by the linker. When they are not bonded, 50 ≦ M2 / M1 ≦ 200 is preferable, 100 ≦ M2 / M1 ≦ 200 is more preferable, and 150 ≦ M2 / M1 ≦ 200 is more preferable. If it is the said structure, while being able to raise the detection sensitivity of the insulin which exists in a solution, the quantity of the insulin which exists in a solution can be quantified more accurately.

  The fluorescence detection step may include a concentration calculation step of calculating the concentration of insulin contained in the sample from the emission value at the detected fluorescence peak wavelength. More specifically, the fluorescence detection step includes a concentration calculation step of calculating the concentration of insulin contained in the sample from the emission value at the peak wavelength of the detected fluorescence and a calibration curve prepared in advance. Also good. Hereinafter, an example of the concentration calculation step will be described, but the present invention is not limited to this example.

  First, a calibration curve is created prior to the fluorescence detection step.

  Specifically, a plurality of types of sample solutions having different concentrations of insulin, including known concentrations of insulin, the first complex, and the second complex, are prepared. And about each sample solution, the light emission value in the peak wavelength of the fluorescence generate | occur | produced by the light emission resonance energy transfer which arises between a luminescent substance and a fluorescent substance, and the luminescence value in the peak wavelength of the light which a luminescent substance emits are measured.

Next, according to the following calculation formula (1), BRET Unit, which is an index of BRET efficiency, is calculated for each sample solution;
[(Emission value at peak wavelength of fluorescence generated by emission resonance energy transfer) − (Emission value at peak wavelength of light emitted from luminescent substance) × Cf] / (Emission value at peak wavelength of light emitted from luminescent substance) .... Calculation formula (1)
However, Cf was obtained using only the first complex containing the luminescent substance or the second complex containing the luminescent substance under the same experimental conditions (the fluorescence generated by the luminescence resonance energy transfer). Emission value at peak wavelength) / (Emission value at peak wavelength of light emitted by the luminescent substance)

  A calibration curve can then be created on the XY coordinates by plotting the known insulin concentration on the X axis and the calculated BRET Unit on the Y axis.

  In the fluorescence detection step, luminescence resonance energy transfer occurs between a luminescent substance and a fluorescent substance from a solution containing a sample containing an unknown concentration of insulin, a first complex, and a second complex. The generated fluorescence is detected. Then, BRET Unit is calculated by substituting the detected data into the above-described calculation formula (1). Then, the concentration of insulin contained in the sample can be determined by substituting the value of the BRET Unit into the calibration curve.

[2. Insulin detection kit)
The insulin detection kit of the present embodiment is a first polypeptide comprising a luminescent substance or a fluorescent substance linked to a first polypeptide containing an α-CT segment of an insulin receptor and not containing a β subunit of the insulin receptor. And a second complex in which a luminescent substance or a fluorescent substance is linked to a second polypeptide that contains the L1 domain of the insulin receptor and does not contain the β subunit of the insulin receptor. It is what.

  Furthermore, in the insulin detection kit of the present embodiment, the luminescent substance and the fluorescent substance generate fluorescence by luminescence resonance energy transfer that occurs between the luminescent substance and the fluorescent substance.

  Furthermore, in the insulin detection kit of the present embodiment, when a luminescent substance is linked to the first polypeptide, a fluorescent substance is linked to the second polypeptide, and the first polypeptide When a fluorescent substance is linked to the polypeptide, a luminescent substance is linked to the second polypeptide.

  In the case where the first complex is a polypeptide, the first complex provided in the insulin detection kit is an expression vector into which a polynucleotide encoding the first complex is inserted in a transcribable manner. It may be a form. Similarly, when the second complex is a polypeptide, the second complex provided in the insulin detection kit has a polynucleotide encoding the second complex inserted in a transcribable manner. It may be in the form of a different expression vector.

  In the insulin detection kit of the present embodiment, the first complex and the second complex may be bonded to each other by a linker to form a third complex. Good.

  When the first complex, the linker, and the second complex are polypeptides (in other words, the third complex includes the first complex, the linker, and the second complex) The insulin detection kit may be provided with an expression vector into which a polynucleotide encoding the third complex is inserted in a transcribable manner.

  The insulin detection kit of the present embodiment may include a support (for example, a glass substrate, a bead, a gel, a cell, or an extracellular matrix) on which the third complex is bound to the surface.

  When the first complex, the linker, the second complex, and the tag are polypeptides (in other words, the first complex, the linker, the second complex, and the tag are In the case of forming one fusion protein), the insulin detection kit may be provided with an expression vector into which a polynucleotide encoding the fusion protein is inserted so as to be transcribed.

  Since the details of the first complex, the second complex, the linker, and the tag have already been described, the description thereof is omitted here.

  In this example, when a desired polypeptide is described as “A polypeptide-B polypeptide”, it is intended that the A polypeptide is linked to the amino terminus of the B polypeptide in the desired polypeptide. To do.

<1. Production and purification of fusion protein>
First, a plasmid for expressing “Nluc-αCT” and a plasmid for expressing “L1-YPet” were prepared. A method for preparing these expression plasmids will be described.

  As αCT of this example, αCT encoded by the DNA sequence shown in SEQ ID NO: 1 and having the amino acid sequence shown in SEQ ID NO: 2 was used. On the other hand, as L1 in this example, L1 encoded by the DNA sequence shown in SEQ ID NO: 3 and having the amino acid sequence shown in SEQ ID NO: 4 was used.

  According to a known method, Nluc was encoded by PCR using pNL 1.1 [Nluc] (Promega), which is an expression plasmid of Nluc (DNA sequence: SEQ ID NO: 5, amino acid sequence: SEQ ID NO: 6) as a template. The DNA sequence was amplified and cloned into an expression plasmid for animal cells (pcDNA3.1 / myc-His B (Life Technologies)).

  Next, the DNA sequence shown in SEQ ID NO: 1 was inserted in frame at the 3 ′ end of the DNA sequence encoding Nluc cloned in the expression plasmid for animal cells, and the fusion protein “Nluc” A plasmid for expressing “-αCT” was prepared. Note that “Nluc-αCT” expressed by the plasmid includes an extracellular secretion signal (DNA sequence: SEQ ID NO: 9, amino acid sequence: SEQ ID NO: 10), and His tag (DNA sequence: SEQ ID NO :) for easy purification. 11, amino acid sequence: SEQ ID NO: 12) are linked. Most of the extracellular secretion signal is degraded and removed during the purification process of the fusion protein “Nluc-αCT”.

  Further, according to a known method, YPet is obtained by PCR using an untargeted Aurora B FRET sensor Kif2 substrate (Addgene plasmid 45215), which is an expression plasmid of YPet (DNA sequence: SEQ ID NO: 7, amino acid sequence: SEQ ID NO: 8) as a template. The DNA sequence encoding was amplified and cloned into an expression plasmid for animal cells (pcDNA3.1 / myc-His B (Life Technologies)).

  Next, the DNA sequence shown in SEQ ID NO: 3 was inserted in frame to the 5 ′ end of the DNA sequence encoding YPet cloned into the expression plasmid for animal cells, and the fusion protein “L1 A plasmid for expressing “-Pet” was prepared. Note that “L1-YPet” expressed by the plasmid includes an extracellular secretion signal (DNA sequence: SEQ ID NO: 9, amino acid sequence: SEQ ID NO: 10) and His tag (DNA sequence: SEQ ID NO :) for easy purification. 11, amino acid sequence: SEQ ID NO: 12) are linked. Most of the extracellular secretion signal is degraded and removed during the purification process of the fusion protein “Nluc-αCT”.

  Next, “Nluc-αCT” and “L1-YPet” were expressed using the above-described plasmid and Expi293 Expression System (Life Technologies). Each protein is released into the medium used for culture by a secretion signal, and the secretion signal portion is cleaved.

First, according to the manual, Exp293 cells (7.5 × 10 ⁷ cells) in culture using 30 mL of a medium were transfected with each plasmid.

  Seven days after the transfection, the culture supernatant was collected. The supernatant was dialyzed against a buffer for histidine tag (His-tag) affinity chromatography (20 mM sodium phosphate, 500 mM sodium chloride, 5 mM imidazole, pH 7.4), and then the supernatant after dialysis was treated with His-Trap EF. The target protein (specifically, “Nluc-αCT” and “L1-YPet”) contained in the supernatant after dialysis was adsorbed to a His-Trap EF column. Thereafter, the target protein was eluted from the His-Trap FF column by the imidazole concentration gradient method.

  The obtained eluate was dialyzed against Tris-HCl buffer (25 mM Tris-HCl, 137 mM sodium chloride, pH 8.0) or KREBS-Ringer buffer (Sigma-Aldrich).

  After determining the concentration of the protein contained in the eluate after dialysis using Micro BCA Kit (Thermo Scientific), the eluate after dialysis (per lane) corresponding to 200 ng of protein was treated with 12% polyacrylamide. The sample was subjected to SDS-PAGE using a gel, and the purity of the target protein contained in the eluate was confirmed.

  FIG. 1 is a photograph showing the result of dyeing a polyacrylamide gel after SDS-PAGE using Quick-CBB PLUS (Wako). As shown in FIG. 1, “Nluc-αCT” and “L1-YPet” could be purified with high purity.

  In addition, “αCT-Nluc” and “YPet-L1” were prepared according to the same method as described above, but the obtained “Nluc-αCT” and “L1-YPet” could be obtained in comparison with the obtained amounts. The amounts of “αCT-Nluc” and “YPet-L1” were small (not shown).

<2. Study on Concentration of First Complex and Concentration of Second Complex>
In KREBS-Ringer Buffer, “Nluc-αCT” (final concentration 0.5 μM), “L1-YPet” (final concentrations 0 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 30 μM, 50 μM, 50 μM, 100 μM, or 150 μM) and insulin (Life Technologies, final concentration 10 μM) were mixed to a total of 100 μL and allowed to stand at 25 ° C. for 30 minutes.

  Thereafter, 1 μL of Nano-Glo Luciferase Assay Substrate is added to the above-mentioned mixed solution, and immediately, the emission value at a wavelength of 445 nm and the emission value at a wavelength of 528 nm are measured with a microplate reader (Spectra-Max M5, Molecular Devices Japan, Tokyo, Japan). ).

  BRET Unit, which is an index of BRET efficiency, was calculated using the following calculation formula (2) based on the measured luminescence value.

[(Emission value at a wavelength of 528 nm) − (Emission value at a wavelength of 445 nm) × Cf] / (Emission value at a wavelength of 445 nm)... (2)
However, Cf is a value of (emission value at a wavelength of 528 nm) / (emission value at a wavelength of 445 nm) obtained using only “Nluc-αCT” under the same experimental conditions.

  FIG. 2 shows the test results. From the viewpoint of increasing the detection sensitivity of insulin present in the solution and quantifying the amount of insulin present in the solution more accurately, the concentration of “Nluc-αCT” is M1, and “L1-YPet” is M2. It was found that 50 ≦ M2 / M1 ≦ 200 is preferable, 100 ≦ M2 / M1 ≦ 200 is more preferable, 150 ≦ M2 / M1 ≦ 200 is further preferable, and M2 / M1 = 200 is most preferable.

<3. Determination of insulin>
In KREBS-Ringer Buffer, mix “Nluc-αCT” (final concentration 0.5 μM), “L1-YPet” (final concentration 100 μM), and insulin (Life Technologies, various final concentrations) to a total of 100 μL. And left at 25 ° C. for 30 minutes.

  Thereafter, 1 μL of Nano-Glo Luciferase Assay Substrate (Promega) was added to the above-mentioned mixed solution, and immediately, the emission value at a wavelength of 445 nm and the emission value at a wavelength of 528 nm were measured with a microplate reader (Spectra-Max M5, Molecular Devices Japan, Tokyo, Japan).

  BRET Unit, which is an index of BRET efficiency, was calculated using the above-described calculation formula (2) based on the measured luminescence value.

  FIG. 3 shows the test results. As shown in FIG. 3, it became clear that insulin can be accurately quantified according to the present invention.

  For example, if the BRET efficiency is calculated based on an actual measurement value of a sample whose insulin concentration is unknown, and if the BRET efficiency value is applied to the calibration curve illustrated in FIG. Can be determined.

  In addition, the detection limit concentration of insulin in this test was 800 nM.

<4. Detection of insulin secreted from the living body>
Mouse insulinoma MIN6 cells (see Non-Patent Documents 3 and 4, etc.) were treated with 10% (v / v) Heat-inactivated fetal bovine serum (Thermo Scientific), 0.1 mM 2-mercaptoethanol (Wako, Osaka, Japan). The cells were cultured in dulbecco's modified eagle's medium (Life Technologies) containing 100 units / mL penicillin (Life Technologies) and 100 μg / mL streptomycin (Life Technologies) under conditions of 5% CO ₂ and 37 ° C.

After collecting 1 × 10 ⁷ cells of MIN6 cells in flat culture by trypsin treatment, the MIN6 cells were washed with KREBS-Ringer Buffer. MIN6 cells after washing suspended in KREBS-Ringer Buffer were mixed with “Nluc-αCT” (final concentration 0.5 μM) and “L1-YPet” (final concentration 100 μM).

  After adding 2.5 μL of Nano-Glo Luciferase Assay Substrate (Promega) to the above mixture, (i) glucose (final concentrations 0 mM, 3 mM, 5 mM, 8 mM, 10 mM, 15 mM) was added to the mixture. Or KREBS-Ringer Buffer containing 25 mM), (ii) KREBS-Ringer Buffer containing tolbutamide (final concentration 3 μM), or (iii) KREBS containing diazoxide (final concentration 10 μM) and glucose (final concentration 25 mM) -Ringer Buffer was mixed to a total volume of 50 μL. “Tolbutamide” is a reagent that promotes insulin secretion, and “diazoxide” is a reagent that suppresses insulin secretion.

  Immediately thereafter, an emission value at a wavelength of 445 nm and an emission value at a wavelength of 528 nm were measured every 10 seconds with a microplate reader (Spectra-Max M5, Molecular Devices Japan, Tokyo, Japan).

  BRET Unit, which is an index of BRET efficiency, was calculated using the above-described calculation formula (2) based on the measured luminescence value.

  FIG. 4 shows the test results. As shown in FIG. 4, it was clarified that the present invention can detect even insulin secreted by a living body.

<5. Tests using various luminescent materials and various fluorescent materials>
Based on Non-Patent Document 1 (John G. Menting et al., “How insulin engages its primary binding site on the insulin receptor” Nature, 10 January 2013, Vol.493, 241) The distance between the α-CT segment and the L1 domain was predicted to be 5 nm.

  The above-mentioned distance of 5 nm can be reproduced by a linker (amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 13)). Then, if the luminescent substance and the fluorescent substance are directly linked via the linker, the complex when the first complex and the second complex of the present invention form a complex via insulin. The state of the luminescent substance and the fluorescent substance in can be reproduced.

  Thus, whether or not fluorescence is generated by luminescence resonance energy transfer when various luminescent materials and various fluorescent materials are directly connected via the linker was examined. Specific test methods and test results will be described below.

  In this example, a luminescent protein (specifically, Nluc) was used as the luminescent substance, and a fluorescent protein (specifically, EYFP, Venus, Ypet) was used as the fluorescent substance.

First, according to methods well known in the six fusion proteins (specifically, "Nluc- _(GGGGS) 3 -EYFP", "Nluc- _(GGGGS) 3 -Venus", "Nluc- _(GGGGS) 3 -YPet "," EYFP- _(GGGGS) 3 -Nluc "," Venus- _(GGGGS) 3 -Nluc "to prepare an expression vector for expressing each of the" YPet- _(GGGGS) 3 -Nluc ").

CHO-K1 cells were treated with Ham's F12 containing 10% (v / v) Heat-inactivated fetal bovine serum (Thermo Scientific), 100 units / mL penicillin (Life Technologies), and 100 μg / mL streptomycin (Life Technologies). The cells were cultured in Nutrient Mixture (Life Technologies) under the conditions of 5% CO ₂ and 37 ° C.

1 × 10 ⁶ cells of CHO-K1 cells were seeded in a 3.5 cm dish, and the next day, 2.5 μg of each expression vector was transfected into CHO-K1 cells using Lipofectamin LTX (Life Technologies).

  48 hours after the transfection operation, CHO-K1 cells were disrupted and luminescence was measured. Specifically, Nano-Glo Luciferase Assay Buffer (Promega) diluted twice with PBS was used as a lysis solution for disrupting CHO-K1 cells. The transfected CHO-K1 cells were washed twice with PBS, and then the supernatant was discarded to prepare a CHO-K1 cell pellet. 200 μL of the lysis solution was added to the pellet, and the well-suspended solution was collected as a cell lysis solution. Add 100 μL of cell lysate to a 96-well plate, mix 5 μL of Nano-Glo Luciferase Assay Substrate (Promega) with the cell lysate, and immediately insert a microplate reader (Spectra-Max M5, Molecular Devices Japan, Tokyo, Japan). The emission spectrum from 400 nm to 600 nm was measured.

  The test results are shown in FIGS. The data shown in FIG. 5 to FIG. 7 are data obtained by standardizing data of actually measured luminescence values by the luminescence value of light emitted from the luminescent material (luminescence value at a wavelength of 445 nm). More specifically, in the data shown in FIGS. 5 to 7, the light emission value near the wavelength 445 nm is the light emission value of the light emitted from the light emitting material, and the light emission value near the wavelength 528 nm is between the light emitting material and the fluorescent material. Is the emission value of the fluorescence generated by the emission resonance energy transfer that occurs in

  As apparent from FIGS. 5 to 7, it has been clarified that the specific configurations of the light emitting substance and the fluorescent substance used in the present invention are not limited. Furthermore, from the viewpoint of increasing the amount of fluorescence emitted by the emission resonance energy transfer, and by increasing the difference between the wavelength of the light emitted by the luminescent material and the wavelength of the fluorescence generated by the emission resonance energy transfer, the fluorescence can be improved. From the viewpoint of measuring the light emission, it has become clear that a combination in which the luminescent substance is Nluc and the fluorescent substance is Venus is preferable, and a combination in which the luminescent substance is Nluc and the fluorescent substance is YPet is more preferable.

  In addition, from the viewpoint of increasing the amount of fluorescence emitted by luminescence resonance energy transfer, the first complex may be one in which a luminescent substance or a fluorescent substance is linked to the amino terminus of the first polypeptide. It has been found that the second complex is preferably one in which a luminescent substance or a fluorescent substance is linked to the carboxyl terminus of the second polypeptide.

<6. Test on Linker Linking First Complex and Second Complex>
<6-1. Production of fusion protein>
First, the first complex and the second complex are converted into various linkers (specifically, AS (SAGG) ₆ SAGSGGLE (amino acid sequence: SEQ ID NO: 14) or AS (GGGGS) ₃ LE (amino acid). A plasmid for expressing the polypeptide linked via the sequence: SEQ ID NO: 25)) was prepared.

Specifically, a plasmid for expressing “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet” (amino acid sequence: SEQ ID NO: 15, DNA sequence: SEQ ID NO: 16), “Nluc-L1-AS ( “SAGG) ₆ SAGSGGLE-αCT-YPet” (amino acid sequence: SEQ ID NO: 17, DNA sequence: SEQ ID NO: 18), “Nluc-αCT-AS (GGGGS) ₃ LE-L1-YPet” (amino acid sequence) : SEQ ID NO: 19, DNA sequence: SEQ ID NO: 20), and "Nluc-L1-AS (GGGGS) ₃ LE-αCT-YPet" (amino acid sequence: SEQ ID NO: 21, DNA sequence: SEQ ID NO: A plasmid for expressing 22) was prepared.

“Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet”, “Nluc-L1-AS (SAGG) ₆ SAGSGGLE-αCT-YPet”, “Nluc-αCT-AS (GGGGS) ₃ LE-L1- YPet ”and“ Nluc-L1-AS (GGGGGS) ₃ LE-αCT-YPet ”have an extracellular secretion signal linked to the amino terminus and Myc tag and His tag linked to the carboxyl terminus. Yes.

The plasmid preparation method is basically the same as that described in <1. Production of the fusion protein and purification> The method described in the above and the well-known method were followed. In addition, <1. Preparation and Purification of Fusion Protein> According to the method described in “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet” and “Nluc-L1-AS (SAGG) ₆ SAGSGGLE-αCT-YPet” Made and purified. In addition, <1. According to the method described in <Production and purification of fusion protein>, “Nluc-αCT-AS (GGGGS) ₃ LE-L1-YPet” and “Nluc-L1-AS (GGGGGS) ₃ LE-αCT-YPet” These were made and were not purified.

  The protein described above was subjected to SDS-PAGE using 12% polyacrylamide gel, and the purity of the protein was confirmed.

FIG. 9 is a photograph showing a part of the result of dyeing a polyacrylamide gel after SDS-PAGE using Quick-CBB PLUS (Wako). More specifically, FIG. 9A shows “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet” (lane 1 in FIG. 9A) and “Nluc-L1-AS” before purification. (SAGG) ₆ SAGSGGLE-αCT-YPet ”(lane 2 in FIG. 9A) shows the test results, and FIG. 9B shows“ Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1- after purification. The test results of “YPet” (lane 3 in FIG. 9B) and “Nluc-L1-AS (SAGG) ₆ SAGSGGLE-αCT-YPet” (lane 4 in FIG. 9B) are shown.

In this test, each of “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet” and “Nluc-L1-AS (SAGG) ₆ SAGSGGLE-αCT-YPet” could be purified with high purity. .

<6-2. Determination of insulin>
“Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet”, “Nluc-αCT-AS (GGGGS) ₃ LE-L1-YPet”, and “Nluc-L1-AS (GGGGS) ₃ LE-αCT-” Each of “YPet” was used to quantify insulin. The test method and test results will be described below.

(I) Test method of “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet”
“Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet” (final concentration 50 nM) and insulin (Life Technologies, various final concentrations) in Tris-HCl buffer (Ph8.0) (+ BSA) Were mixed to a total of 100 μL and allowed to stand at 25 ° C. for 20 minutes.

  Thereafter, 1 μL of Nano-Glo Luciferase Assay Substrate (Promega) is added to the above-mentioned mixed solution and mixed. Measurement was performed with a plate reader (Spectra-Max M5, Molecular Devices Japan, Tokyo, Japan).

(Ii) Test method for “Nluc-αCT-AS (GGGGGS) ₃ LE-L1-YPet” and “Nluc-L1-AS (GGGGS) ₃ LE-αCT-YPet” <5. The test was performed according to the method described in the> Tests Using Various Luminescent Substances and Various Fluorescent Substances>, and emission spectra from 400 nm to 600 nm were measured.

  BRET Unit, which is an index of BRET efficiency, was calculated using the following calculation formula (3) based on the measured luminescence value.

[(Emission value at a wavelength of 528 nm) − (Emission value at a wavelength of 445 nm) × Cf] / (Emission value at a wavelength of 445 nm)... (3)
However, Cf was a value obtained by using only “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet” under the same experimental conditions (emission value at a wavelength of 528 nm) / (emission value at a wavelength of 445 nm). It is.

10 and 11 show the test results of “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet”. As shown in FIG. 10, if “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet”, depending on the concentration of insulin, the emission value at a wavelength of 528 nm increases with respect to the emission value at a wavelength of 445 nm, In other words, it has become clear that insulin can be accurately quantified. Moreover, as shown in FIG. 11, the insulin detection limit concentration when “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet” was used was 50 nM. On the other hand, <3. As already described in the column “quantification of insulin>, the insulin detection limit concentration in the case where the first complex and the second complex are not linked by a linker was 800 nM. That is, when the first complex and the second complex are linked by a linker, the sensitivity of insulin detection is higher than when the first complex and the second complex are not linked by a linker. It became clear that it improved dramatically.

FIG. 12 shows the test results of “Nluc-αCT-AS (GGGGS) ₃ LE-L1-YPet”. As shown in FIG. 12, with “Nluc-αCT-AS (GGGGS) ₃ LE-L1-YPet”, the emission value at a wavelength of 528 nm may increase with respect to the emission value at a wavelength of 445 nm depending on the presence of insulin. It was revealed. Moreover, in the case of “Nluc-αCT-AS (GGGGS) ₃ LE-L1-YPet”, compared with the case of “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet”, the same insulin concentration It became clear that the value of the luminescence value was low (in other words, the detection sensitivity of insulin was low).

FIG. 13 shows the test results of “Nluc-L1-AS (GGGGS) ₃ LE-αCT-YPet”. As shown in FIG. 13, in the case of “Nluc-L1-AS (GGGGGS) ₃ LE-αCT-YPet”, it was revealed that the luminescence value does not increase even when insulin is present at a high concentration. In the case of “Nluc-L1-AS (SAGG) ₆ SAGSGGLE-αCT-YPet”, similarly to “Nluc-L1-AS (GGGGS) ₃ LE-αCT-YPet”, even if a high concentration of insulin exists. The light emission value tended to be difficult to increase (not shown).

<7. Sensor cell>
<7-1. Production of sensor cells>
In the above-mentioned plasmid for expressing “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet”, the transmembrane region of PDGF (platelet-derived growth factor) receptor (hereinafter, referred to as “platelet-derived growth factor” receptor) DNA encoding TMR (also referred to as trans-membrane region) is inserted in-frame, and “Nluc-αCT-AS (SAGG) ₆ SAGSGGLE-L1-YPet-TMR” (amino acid sequence: SEQ ID NO: 23, DNA A plasmid for expressing the sequence: SEQ ID NO: 24) was prepared. After the plasmid was linearized with a restriction enzyme, the plasmid was purified according to a conventional method.

Hepa1-6 cells were seeded in a 24 well dish at a concentration of 5 × 10 ⁵ cells / well.

  (I) A solution in which 500 ng of linearized plasmid and DMEM medium were mixed (total 25 μL), and (ii) a solution in which 2 μL of Lipofectamine LTX (Lifetechnologies) and DMEM medium were mixed (total 25 μL) were prepared. did.

  The solutions (i) and (ii) described above were mixed, and the mixed solution was incubated at room temperature (25 ° C.) for 20 minutes.

  After washing Hepa1-6 cells twice with PBS, 450 μL of DMEM medium and 50 μL of the above mixed solution were added to the Hepa1-6 cells. Subsequently, the Hepa1-6 cells were cultured at 37 ° C. for 48 hours.

  Thereafter, Hepa1-6 cells were cultured in a DMEM medium containing FBS (10%) and G418 (1000 μg / mL) for 1 week or longer to obtain a stably transformed Hepa1-6 cell line.

<7-2. Imaging of insulin using sensor cells>
100 μL of a 1N NaOH solution was added to a glass base dish (Matsunami 35 mm multiwell glass bottom dish, diameter 9.5 mm, 4 well). The glass base dish was incubated at room temperature (25 ° C.) for 30 minutes, and then washed three times with PBS, so that the cell adhesion surface of the glass base dish was made hydrophilic. Next, 100 μL of PBS containing gelatin Type B at a concentration of 0.1% was added to the glass base dish, and the glass base dish was incubated at 37 ° C. for 10 minutes. Thereafter, the glass base dish was washed once with PBS.

After washing MIN6 cells (insulin-secreting cells) cultured by a conventional method with PBS, the MIN6 cells were stained with CellTracker ^™ Blue CMF2HC Dye according to the manual. Each of the sensor cells and MIN6 cells was detached from the culture dish by trypsin treatment, and after mixing each cell by 0.5 × 10 ⁵ cells (total 1 × 10 ⁵ cells / well), all the cells were described above. Glass seeds were seeded and cultured overnight at 37 ° C. and 5% CO ₂ .

  1 μL of Nano-Glo Luciferase Assay Substrate was added to 100 μL of KREBS-Ringer buffer containing glucose (final concentration 0 mM or 25 mM) and mixed well to prepare a reaction solution.

  The mixed culture of sensor cells and MIN6 cells after overnight culture was washed once with PBS, and the reaction solution described above was added to the mixed culture.

The mixed culture is immediately set on a fluorescence microscope, and a sensor cell expressing YPet is identified by fluorescence observation using a filter set for FITC, and CellTracker ^{TM is} observed by fluorescence observation using a filter set for DAPI. MIN6 cells stained with Blue CMF2HC Dye were identified (see FIG. 14).

  Next, the excitation light is blocked, and an image (Nluc image) observed through a DAPI filter and an image (YPet image) observed through a FITC filter are displayed every 10 seconds for 1 hour. , And continuous shooting using a 40 × objective lens. After removing the background from each acquired image, “signal intensity in YPet image / signal intensity in Nluc image” was calculated for each pixel at each time.

  The above-mentioned “Signal intensity in YPet image / Signal intensity in Nluc image” was converted into a color image using HC Image (Hamamatsu Photonics) (see FIG. 15 and FIG. 16).

  Furthermore, the region was set so as to include cells, and the change over time of the average value of the signal intensity of the pixels included in the region was plotted (see FIG. 17).

  As apparent from FIGS. 15 to 17, the sensor cell was able to detect insulin secreted by the MIN6 cells.

  If the sensor cell is used, insulin secretion by individual cells can be detected. For example, if the behavior of the sensor cell is observed in a state where one cell is surrounded by the sensor cell, insulin secretion by the one cell can be detected.

  When the sensor cell is used, insulin secretion by the target cell can be easily detected by co-culturing the target cell and the sensor cell.

  When the sensor cell is used, insulin secretion in an environment closer to the living body can be detected by three-dimensional co-culture of the target cell and the sensor cell.

  By using the sensor cell, a chimeric mouse containing the sensor cell in the body can be produced. And if the said chimeric mouse | mouth is used, the secretion of insulin in the environment close | similar to a biological body is detectable.

  INDUSTRIAL APPLICABILITY The present invention can be widely used in the pharmaceutical field, medical device field, and the like where it is necessary to detect the presence of insulin and / or measure the concentration of insulin.

Claims (9)

A sample, a first complex comprising a luminescent substance or a fluorescent substance linked to a first polypeptide containing an insulin receptor α-CT segment and not containing an insulin receptor β subunit; From a solution comprising a second complex containing a L1 domain and not containing an insulin receptor β subunit and a second complex in which a luminescent substance or a fluorescent substance is linked, the luminescent substance and the fluorescent substance A fluorescence detection step of detecting fluorescence generated by emission resonance energy transfer occurring between the substance and
When a luminescent substance is linked to the first polypeptide, a fluorescent substance is linked to the second polypeptide, and when a fluorescent substance is linked to the first polypeptide, A method for detecting insulin, wherein a luminescent substance is linked to the second polypeptide.   2. The concentration (M1) of the first complex and the concentration (M2) of the second complex in the solution satisfy a relationship of 50 ≦ M2 / M1 ≦ 200. The method for detecting insulin according to 1.   The method for detecting insulin according to claim 1 or 2, wherein the first complex is obtained by linking the luminescent substance or the fluorescent substance to the amino terminus of the first polypeptide.   The said 2nd composite_body | complex is what said luminescent substance or said fluorescent substance was connected with the carboxyl terminus of said 2nd polypeptide, The any one of Claims 1-3 characterized by the above-mentioned. Insulin detection method.   The insulin according to claim 1, 3 or 4, wherein the first complex and the second complex are bonded to each other by a linker to form a third complex. Detection method.   6. The method for detecting insulin according to claim 5, wherein the third complex is bound to the surface of the support. A first complex comprising a luminescent substance or a fluorescent substance linked to a first polypeptide containing the α-CT segment of the insulin receptor and not containing the β subunit of the insulin receptor, and the L1 domain of the insulin receptor. A second complex containing a luminescent substance or a fluorescent substance and a second polypeptide containing and not containing the β subunit of the insulin receptor,
The luminescent substance and the fluorescent substance generate fluorescence by luminescence resonance energy transfer that occurs between the luminescent substance and the fluorescent substance,
When a luminescent substance is linked to the first polypeptide, a fluorescent substance is linked to the second polypeptide, and when a fluorescent substance is linked to the first polypeptide, A kit for detecting insulin, wherein a luminescent substance is linked to the second polypeptide.   The insulin detection kit according to claim 7, wherein the first complex and the second complex are bonded to each other by a linker to form a third complex.   9. The insulin detection kit according to claim 8, wherein the insulin detection kit includes a support body on which the third complex is bound to the surface.